Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of se...Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.展开更多
DNA double-strand breaks（DSBs）,which arise following exposure to a number of endogenous and exogenous agents,can be repaired by either the homologous recombination（HR）or non-homologous end-joining（NHEJ） pathways...DNA double-strand breaks（DSBs）,which arise following exposure to a number of endogenous and exogenous agents,can be repaired by either the homologous recombination（HR）or non-homologous end-joining（NHEJ） pathways in eukaryotic cells.A vital step in HR repair is DNA end resection,which generates a long 30single-stranded DNA（ss DNA） tail that can invade the homologous DNA strand.The generation of 30 ss DNA is not only essential for HR repair,but also promotes activation of the ataxia telangiectasia and Rad3-related protein（ATR）.Multiple factors,including the MRN/X complex,C-terminal-binding protein interacting protein（Ct IP）/Sae2,exonuclease 1（EXO1）,Bloom syndrome protein（BLM）/Sgs1,DNA2 nuclease/helicase,and several chromatin remodelers,cooperate to complete the process of end resection.Here we review the basic machinery involved in DNA end resection in eukaryotic cells.展开更多
基金provided by the grant(2013-33522-21091 to B.Y.) from the USDA Biotechnology Risk Assessment program
文摘Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.
基金supported in part by the grants from the National Natural Science Foundation of China (Grant Nos.31071243 and 31171347)the Fundamental Research Funds for the Central Universities of Chinathe Research Fund for the Doctoral Program of Higher Education of China (Grant No.20110101120152)
文摘DNA double-strand breaks（DSBs）,which arise following exposure to a number of endogenous and exogenous agents,can be repaired by either the homologous recombination（HR）or non-homologous end-joining（NHEJ） pathways in eukaryotic cells.A vital step in HR repair is DNA end resection,which generates a long 30single-stranded DNA（ss DNA） tail that can invade the homologous DNA strand.The generation of 30 ss DNA is not only essential for HR repair,but also promotes activation of the ataxia telangiectasia and Rad3-related protein（ATR）.Multiple factors,including the MRN/X complex,C-terminal-binding protein interacting protein（Ct IP）/Sae2,exonuclease 1（EXO1）,Bloom syndrome protein（BLM）/Sgs1,DNA2 nuclease/helicase,and several chromatin remodelers,cooperate to complete the process of end resection.Here we review the basic machinery involved in DNA end resection in eukaryotic cells.
